Aorta. The level of mRNA of ryanodine (RyR2) and Inositol-1,4,5-trisphosphate (IP3R1, IP3R2, IP3R3) receptors as well as regulatory proteins – CaM, Epac1 and Epac2 - were evaluated in the aortic tissues of male and female rats. Analysis of the results shows that the aorta of old rats of both sexes (24 months) decreases the level of mRNA for IP3R2 (up to 63% and 61%, respectively in females and males) and IP3R3 (up to 62% and 67%, respectively in females and males) and doesn't change the expression of IP3R2-type receptors in comparison with similar data in young rats (4 months). It was found that in old rats regardless of the sex of animals the content of mRNA for RyR2 in blood vessels increased by more than 1,5 times (up to 153% in males and up to 185% in females) (Fig. 1a).
However, there are significant sexual discrepancies in nature of age-related changes in the expression of CaM genes and Epac proteins. It was proven that in old females the level of mRNA in the aorta decreases for CaM and Epac1 (by 30% and 32%, respectively) and doesn't change for Epac2 in comparison with similar indicators in the vessels of young females (Fig.1b). In contrast to females, the expression of Epac1 and Epac2 proteins increases in the vessels of old male rats and doesn't change for CaM (Fig. 1b).
IP3Rs function in vessels.
IP3Rs play a central role in generating and maintaining myogenic vascular tone. However, it is still unknown exactly what contribution each of the three IP3Rs subtypes makes to the complex transmission of Ca2+ signals in the cell. The role of IP3R vessels in the development of hypertension remains unclear and data available rests contradictory. It is supposed that in hypertension increased sensitivity to phenylephrine is caused by an increase in the expression of IP3R1 in vascular SMCs as a result of Ca2+ - dependent activation of the transcription factor CN-NFAT (calcineurin-Nuclear factor of activated T-cells) . At the same time, activation of IP3R1 in cerebral arteries located in close proximity to Ca2+-activated high-conductivity potassium channels (BKCa) has been shown to increase the sensitivity of these channels to Ca2+. According to the authors, this mechanism can limit IP3-induced vasoconstriction in the cerebral arteries .
Experiments on HeLa cell culture that express comparable amounts of IP3R1 and IP3R3 showed that suppression of IP3R1 subtype expression by RNA interference leads to a significant decrease in total Ca2+ signals and the termination of Ca2+ oscillations . In contrast, suppression of IP3R3 expression leads to more stable and prolonged fluctuations in intracellular Ca2+ concentration than in the control. Similar effects of IP3R3 knockout were obtained on COS-7 cells, which primarily Express IP3R3 . These results allowed the authors to suggest that IP3R1 and IP3R3 cells have opposite effects on the generation of intracellular Ca2+ oscillations.
Experiments on rat aortic SMCs culture showed that proliferating cells express mainly IP3R2 and IP3R3 but during their development the expression of these types of receptors decreases, and IP3R1, on the contrary, increases [36, 37]. It was shown that IP3R1 is distributed throughout the cytoplasm, IP3R2 is closely related to the nucleus and to the plasma membrane, and IP3R3 is distributed mainly around the nucleus. It is shown that each isoform of IP3Rs can lead to activation of apoptosis . However, the IP3R3 isoform plays a special role in inducing apoptosis primarily by transmitting Ca2+ signals to mitochondria, since IP3R3 has a special tendency to form subcellular clusters with mitochondria, which leads to the formation of associated signaling microdomains. Interestingly, IP3R3 can not only activate, but also inhibit apoptosis, and this process is implemented by an Akt-IP3R3-dependent mechanism .
We determined that one of the manifestations of age-related vascular changes is an imbalance in the expression of IP3Rs: the expression of type 2 and 3 receptors decreases and IP3R1 doesn't change. These changes in the expression of IP3Rs subtypes in aging vessels of animals of both sexes, apparently, can be critical for various forms of Ca2+ signal transmission in both SMCs and endothelial cells and affect the contractile properties of vessels. In addition, it can be assumed that a significant decrease in the expression of IP3R2 and IP3R3 types in the aorta of old rats of both sexes is a manifestation of an age-related decrease in the proliferative and apoptotic activity of SMCs.
RyRs function in vessels.
RyRs function in SMC is less clear. Although RyRs in smooth muscles generate local Ca2+sparks that are similar to those observed in striated muscles, however, the contribution of these changes to the depolarization-induced global increase in intracellular Ca2+ ([Ca2+]i) concentration is quite limited. Unlike cardiac or skeletal muscles, RyR-dependent Ca2+ - induced calcium release (CICR) isn't necessary for vasoconstriction as the concentration of Ca2+ in vascular SMC causes vascular contraction that is lower than required for RyR-mediated CICR activation . In vascular SMC RyR-mediated Ca2+ sparks are accompanied by activation of BKCa that leads to a decrease in myogenic tone and vasodilation. At the same time, it is shown that BKCa and RyR channels play different roles in the regulation of myogenic and neurogenic tone: while BKCa and RyR channels work together to counteract myogenic vasoconstriction, BKCa channels counteract neurogenic vasoconstriction, and RyR channels, on the contrary, enhance it . The authors suggest that at the level of neurogenic vasoconstriction of resistive arteries, RyRs act in conjunction with voltage-dependent L-type Ca2+ channels.
It was determined that in vessels of old females and males, the level of mRNA RyR2 increases by 1.9 and 1.5 times respectively compared to similar indicators in young rats. An increase in the expression of RyR2 and a decrease in IP3R2 and IP3R3 in the aorta of rats of both sexes caused by aging indicate significant changes in intracellular Ca2+ metabolism but it isn't clear yet how they affect the regulation of myogenic and neurogenic vascular tone in a whole organism. Moreover, the expression of the most important regulators of channel protein activity – CaM and Epac — changes during vascular aging. We found out that within a high expression of RyR2 in the aorta of old females, there is a significant decrease in the expression of CaM and Epac1, while in the vessels of males the level of mRNA for CaM remains unchanged but the expression of Epac1 and Epac2 proteins increases.
For RyRs calcium is the main physiological activity regulator [30, 42]. With a moderate increase in [Ca2+] i the excitation of RyRs is mediated through activation of CaMK-dependent channel phosphorylation or by interaction of Ca2+ with the Ca2+ - binding protein S100A which competes with СaM for binding to RyRs. Vasodilators that act on receptors associated with the Gs protein activate adenylate cyclase, which leads to an increase in cAMP and subsequent activation of PKA- and Epac-mediated phosphorylation of RyRs, which leads to an increase in the frequency of RyR-dependent local calcium signals, followed by activation of BKCa-channels and vasodilation. At a high level of [Ca2+] i, RyR activity, in contrast, is inhibited through mechanisms including Ca2+-binding sorbin proteins (SORC) or CaM .
Thus, considering aging, unidirectional changes in the expression of genes for channel proteins IP3Rs and RyR2 develop in the aorta of rats, and changes in the expression of proteins that modulate their activity – CaM and Epac indicating age-related changes in intracellular calcium metabolism. It can be assumed that a high level of Epac1 and Epac 2 expression in the vessels of old male rats negatively affects the activity of IP3Rs and positively affects RyRs, since Epac-induced phosphorylation leads to a decrease in the activity of the former and an increase in the latter. A decrease in CaM expression in the vessels of older females may lead to a decrease in its inhibitory effect on IP3Rs and RyRs, and Epac1 may lead to a decrease in its activating effect on RyR2 and inhibiting on IP3Rs. Apparently, one of the aging vessels signs is a violation of the goodness of fit modulation mechanisms of the most important ion channels involved in the regulation of [Ca2+]i, which have a certain specificity in female and male rats. The detected changes can make a significant contribution to age-initiated vascular pathology at the level of their contractile, proliferative and apoptotic activity. However, it is possible that some of them may be a manifestation of compensatory mechanisms aimed at preserving Ca2+-homeostasis in aging vessels.
Heart. It was determined that in old male rats, the level of mRNA RyR2 increases in the left ventricle and both atria (Fig. 2). In older males, the content of mRNA in the left ventricle increases for all three types of IP3Rs, in the left atrium only for IP3R1-type, in the right atrium – increases for IP3R3-type, but the level of mRNA for IP3R2-type decreases compared to control animals (Fig. 2).
Other age-related changes in the expression of the RyR2 and IP3Rs genes were detected in the hearts of old females. It was found that the expression of RyR2, IP3R1, and IP3R3 decreases in the left ventricle during aging in females in contrast to males, while the level of mRNA for IP3R3-type increases in the left atrium, and for IP3R1 – and IP3R3 - type receptors in the right atrium (Fig. 2). the level of expression of IP3R2-type mRNA in the hearts of old females does not differ from the indicators in the hearts of young rats.
While aging, the expression of CaM and Epac2 increases significantly in the left ventricle and atria of rats of both sexes as evidenced by a higher level of their mRNA compared to similar indicators in young animals (Fig. 3). In older males, the mRNA level for Epac1 remains unchanged in the heart, while in females, the mRNA level of this protein decreases in the left ventricle and, on the contrary, increases in the left atrium (Fig. 3).
It is known that the aging heart is characterized by a reduced response to sympathetic stimulation, a high risk of arrhythmias and sudden cardiac death [43-45]. Age-related electrical instability and contractile dysfunction of the myocardium are primarily associated with impaired Ca2+-homeostasis. However, specific molecular mechanisms underlying the altered regulation of Ca2+ metabolism in the aging heart remain poorly understood. It is shown that older people have reduced functional activity of the sinoatrial node, that is believed to be associated with an age-related decrease in the expression of the main Ca2+ - conducting channel SR-RyR2, as well as with changes in the expression of other ion channels – an increase in Na(v)1.5, Na(v)β1 and Ca(v)1.2 and a decrease in K(v)1.5 and HCN1 .
RyRs function in heart.
It is supposed that RyR2 receptors are the key intracellular structures that implement the effect of the sympathetic nervous system on the myocardium. Our research has shown that in older males, the expression of RyR2 increases significantly in the left ventricle and atria, while in females, on the contrary, it decreases in the left ventricle and doesn't change in the atria. Along with a change in the expression of RyR2 in the aging hearts of rats of both sexes, a high level of expression of the mRNA proteins CaM and Epac2 was detected. Changes in the transcription level of these genes can lead to excessive activation of RyR2 and to serious violations of calcium metabolism in cardiomyocytes [25, 47]. It was found that an excessive increase in the activity of RyR2 as a result of their oxidation, nitrosylation or hyperphosphorylation contributes to the development of heart failure and the occurrence of ventricular arrhythmias [48, 49]. It is known that activation of β-adrenoreceptors (β-AR) can trigger cardiac arrhythmias through cAMP-dependent mechanisms by increasing Ca2+ leakage from SR to diastole. cAMP can activate both PKA and Epac proteins. In studies using genetic modifications of Epac1, Epac2, RyR2, and CaMKII, it has been shown that activation of Epac and PKA can equally contribute to the occurrence of β-AR-induced arrhythmias and Ca2+ leak from SR. It has been proved that the β1-AR induced increase in arrhythmogenic Ca2+ emissions is mediated by Epac2 . The arrhythmogenic effect of Epac2 has been demonstrated in numerous studies in recent years [47, 51]. It was found that the β-AR-cAMP-Epac2-PI3K-Akt-NOS1-CaMKII signaling cascade mediates increased (pathological) Ca2+ leak from SR to diastole, while the cAMP-PKA pathway mainly implements inotropic and lucitropic effects of β-AR .
In cardiomyocytes, CaM regulates the activity of RyR2 by direct interaction with them, as well as through CaMKII, which phosphorylate both RyR2 and IP3Rs . CaM reduces the probability of opening RyR2 at submicromolar (diastolic) and micromolar (systolic) concentrations of Ca2+. CaM reduces the probability of opening RyR2 at submicromolar (diastolic) and micromolar (systolic) concentrations of Ca2+. Recent years data have been obtained on the key role of CaM in the mechanisms of development of heart hypertrophy. In vitro and in vivo experiments have shown that the suppression of CaM gene expression by microRNA has a pronounced antihypertrophic effect . It was determined that point mutations in the CaM-binding domain of RyR2 (W3587A/ L3591D/F3603A) in mice lead to severe heart hypertrophy, a sharp decrease in left ventricular inotropic function, and early death of animals [55, 56]. Our data on the high level of expression of RyR2, СaM and Epac2 in the hearts of old animals suggest that males are much more likely to develop myocardial hypertrophy and ventricular arrhythmias than females (Fig. 2, 3).
IP3Rs heart functions.
If the role of RyR2 as a receptor regulating the release of Ca2+ from SR, necessary for the implementation of electromechanical coupling of cardiomyocytes is obvious, there is still no clear understanding of IP3Rs contribution to this process. Under physiological conditions cardiomyocytes, in contrast to non-excitable cells, have a low level of Gq-induced IP3 generation and a weak IP3R response . The role of IP3Rs in pathophysiology, especially in arrhythmogenesis and myocardial hypertrophy, is more obvious. Currently, it is proved that in the global increase in [Ca2+]i in cardiomyocytes, the share of IP3R-induced release of Ca2+ from SR is insignificant, which makes their contribution to the regulation of the process of electro-mechanical coupling of cardiomyocytes minimal. It is considered that an important condition for IP3Rs activation is their localization in the subsarcolemmal zone meaning in the immediate vicinity of the IP3 generation site .
This study identifies sexual dimorphisms of age-related changes in the expression level of IP3R1–IP3R3 type receptors in the left ventricle of the hearts of old rats. Thus, in old males, the expression of IP3R1, IP3R2, and IP3R3 in the left ventricle was significantly higher, while in females, on the contrary, it was lower (IP3R1 and IP3R3) than in the hearts of young rats.
IP3R1 is localized in the heart mainly in conducting and IP3R2 in contractile cardiomyocytes and IP3R2 is located mainly in the perinuclear membrane region in ventricular cardiomyocytes [57, 58]. Atrial cells express a greater number of IP3Rs compared to ventricular cardiomyocytes. They are mostly dominated by type 1 and type 2 IP3Rs . In atria the amount of IP3R2 is 6-10 times greater than in ventricles, and in SR they are in close proximity to the sarcolemma in close proximity to the RyRs [57, 58]. It has been shown that agonist-induced activation of phospholipase C (PLC) in atrial cardiomyocytes causes local Ca2+sparks that can be transmitted to nearby RyR2 and amplify the CICR response, and thus IP3Rs can affect the process of electro-mechanical coupling in atria . It is assumed that IP3R-induced activation of RyR2, other ion channels and exchangers, in particular Ca2+ - l-type channels and Na+/Ca2+ - exchanger, can lead to atrial fibrillation .
Taking into account the literature data on the predominant localization of IP3R1 type in conducting cardiomyocytes, it can be assumed that an increase in the expression of this receptor isoform in the left ventricle and left atrium of old male rats is a risk factor for arrhythmias. In addition, a high level of IP3R2 expression in the left atrium can also provoke atrial fibrillation, since their role in arrhythmogenesis has been proven [61, 62].
It was determined that the GPCR/IP3R axis modulates not only heart rhythm disorders but also the hypertrophy development. There is strong evidence that IP3R2 is involved in the development of myocardial hypertrophy under conditions of increased expression of these receptors and/or their over-activation as a result of agonist-induced signaling from GPCR , and these mechanisms are implemented at the level of nuclear membranes.
In recent years, more and more attention has been paid to studying the features of Ca2+ signal transmission in the nuclei of cardiomyocytes and their role in the regulation of gene transcription [64, 65]. The transcription of genes and cell growth is influenced by nuclear Ca2+ signals, which differ from cytosolic Ca2+ signals. The regulation of intranuclear concentration of Ca2+ ([Ca2+]nuc) is carried out mainly through passive diffusion of ions from the cytoplasm. However, there is an additional source of Ca2+ in the nucleus that is completely independent of the cytosolic one: the nuclear envelope (NE), which is a cell compartment with its own perinuclear Ca2+ storage . In NE, proteins that regulate Ca2+ traffic, including IP3R, are expressed that address both the nucleoplasm and the cytoplasm . Specific GPCR stimuli can increase [Ca2+]nuc via IP3R-mediated release of Ca2+ from NE regardless of the concentration of Ca2+ ions in the cell cytosol ([Ca2+]cyt) . In contrast to the global increase in [Ca2+]cyt, it is currently believed that the local increase in [Ca2+]nuc plays a central role in the regulation of gene expression in cardiomyocytes.
The structural organization of Ca2+signal transmission in the nucleus may include both receptors and IP3 generation on the T-channel membranes of ventricular cardiomyocytes located in the immediate vicinity of the nucleus, and GPCR complexes localized on NE [64, 65]. It is known that IP3R2-type ventricular cardiomyocytes are concentrated in NE . Specific GPCR stimuli, such as endothelin 1, angiotensin II, and others, can increase [Ca2+]nuc independently of [Ca2+]cyt through IP3R2-mediated release of Ca2+ from NE . IP3R2 localized on NE are associated with CaM, CaMKII. Activation of CaMKII-dependent nuclear signaling triggers the phosphorylation of class II histone deacetylases (HDAC) and the export of nuclear HDACs, which leads to the activation of MEF2-dependent transcription of various genes (myocyte enhancer factor-2) . The CaN-NFAT transcriptional pathway is also activated. The presence of local Ca2+ -dependent mechanisms of IP3R2-mediated regulation of nuclear pore permeability for transcription factor traffic in adult rat cardiomyocytes suggests that IP3R2 is involved in the development of pathological left ventricular hypertrophy in old males. This assumption is supported by the high level of expression of IP3R2, as well as CaM in cardiomyocytes of the left ventricle of rats at the age of 24 months compared with similar rates in young rats.
In contrast to males, older females are highly likely to develop atrial fibrillation, since the level of IP3R1 expression significantly increases in the right atrium of females during aging. An increase in IP3Rs is also observed in atrial cardiomyocytes in patients with chronic atrial fibrillation [71, 72]. Unlike males, in older females, the risk of developing ventricular arrhythmias, as well as their hypertrophy, is minimal, since they have a low level of expression of RyR2, IP3R1 and IP3R3 in the left ventricle and unchanged for IP3R2 type.
It has been determined that the IP3R3 receptor isoform is involved in the development of various human diseases . However, the role of IP3R3 in the regulation of cardiac activity in normal and pathological conditions has been less studied. It is known that IP3R3-dependent Ca2+-signaling is necessary for NO-induced differentiation of cardiomyocytes obtained from embryonic stem cells . Based on the above data on the important contribution of IP3R3 to the modulation of the generation of Ca2+ oscillations , it can be assumed that a significant increase in the expression of these receptors in cardiomyocytes of rats of both sexes in combination with altered expression of RyR2, IP3R1 and IP3R2 indicates a transition to a new level of regulation of intracellular calcium exchange in the hearts of old rats.